Regulation of acetylation of plant cell wall components is complex and responds to external stimuli

ABSTRACT

Previously, we reported that the allelic de-etiolated by zinc (dez) and trichome birefringence (tbr) mutants exhibit photomorphogenic development in the dark, which is enhanced by high Zn. TRICHOME BIREFRINGENCE-LIKE proteins had been implicated in transferring acetyl groups to various hemicelluloses. Pectin O-acetylation levels were lower in dark-grown dez seedlings than in the wild type. We observed Zn-enhanced photomorphogenesis in the dark also in the reduced wall acetylation 2 (rwa2-3) mutant, which exhibits lowered O-acetylation levels of cell wall macromolecules including pectins and xyloglucans, supporting a role for cell wall macromolecule O-acetylation in the photomorphogenic phenotypes of rwa2-3 and dez. Application of very short oligogalacturonides (vsOGs) restored skotomorphogenesis in dark-grown dez and rwa2-3. Here we demonstrate that in dez, O-acetylation of non-pectin cell wall components, notably of xyloglucan, is enhanced. Our results highlight the complexity of cell wall homeostasis and indicate against an influence of xyloglucan O-acetylation on light-dependent seedling development.

Earlier, we provided evidence supporting a role of vsOGs, very small fragments of pectin, as cell-to-cell signals required for maintaining skotomorphogenesis in the dark.¹ We proposed that the loss of this signal in dez mutant seedlings resulted in a weak photomorphogenic phenotype when germinated in the dark, and this was strongly enhanced by the presence of high Zn in the growth medium.¹ The dez phenotype cannot be attributed to mere growth defects, given that we also observed photosynthetic pigment accumulation and partial chloroplast differentiation in dark-grown dez. Cell walls of dez seedlings were significantly thickened, with alterations in both pectin acetylation and pectin methylesterification.¹ TRICHOME BIREFRINGENCE-LIKE (TBL) proteins share a predicted transmembrane domain, a conserved TBL domain, and a C-terminal PC-esterase domain (PMID: 20056006, previously DUF231).² Previously characterized members of the TBL family were localized in the Golgi where they transfer acetyl groups to varying hemicelluloses including xylan,^3–5 xyloglucan⁶ and mannan.⁷

Reduced Wall Acetylation (RWA) family proteins are thought to transport the substrate acetyl-CoA into the Golgi.^8,9 Accordingly, the acetylation of cell wall polymers is decreased in the rwa2-3 mutant. Interestingly, the rwa2-3 mutant also displayed Zn-dependent photomorphogenesis when grown on high-Zn medium in the dark, but was skotomorphogenic like the wild type under control conditions.^1,8

We observed an extracellular localization of a TBR-GFP fusion protein that complemented the photomorphogenic dez phenotype. In combination with our finding of decreased levels of pectin O-acetylation under both control and high-Zn conditions, our data are in agreement with a previously stated hypothesis that TBR is likely enzymatically inactive and acts to protect O-acetylated pectin from enzymatic de-acetylation in muro.¹⁰ We proposed that the complex changes observed in the cell wall of dez/tbr (see above) reflect the complexity of partly spatially separated and homeostatic processes in the cell wall as demonstrated for pectin methylesterification.^1,10,11 In the presence of high Zn in the dark, there was increased pectin methylesterification but also higher pectin methylesterase (PME) activity in dez suggesting a spatial separation of PME enzyme and its substrate.¹

To globally examine the effect of dez and rwa2-3 mutations, we quantified chemically the levels of O-acetylation of bulk cell wall material, as well as xyloglucan O-acetylation using Oligosaccharide Mass Profiling (OLIMP).^12,13 In 7-day-old dark-grown dez seedlings cultivated in standard medium (control conditions), we observed a 32% increase in the acetylation of bulk cell wall material compared to the wild type (Figure 1(a)). By comparison, acetylation was reduced by 23% in the rwa2-3 mutant, consistent with previously published findings.⁸ When grown on high-Zn medium, the overall acetylation level was lower in dez so that a difference between dez and the wild type was no longer observed, whereas cell wall acetylation was slightly increased to reach wild type-like levels in rwa2-3 (Figure 1(a)).

Figure 1.

Quantification of cell wall acetylation and xyloglucan composition.

(a) Quantification of acetic acid (AC) released from cell wall material (alcohol-insoluble residue, AIR) prepared from 7-d-old dark-grown wild-type (WT), dez and rwa2-3 seedlings grown in control and highZn conditions. (b) Relative abundance of xyloglucan oligosaccharides released by a xyloglucanase from cell wall material of cell wall material of 7-d-old dark-grown seedlings from control medium as determined by OLIMP.¹³ Xyloglucan nomenclature is as previously described.¹¹ X – xylose; G – glucose; L – galactose; F – fucose; OAc – O-acetylated oligosaccharide. (c) Relative abundance of xyloglucan species released from AIR of 7-d-old dark-grown seedlings from highZn medium as determined by OLIMP.¹³ Xyloglucan nomenclature is as previously described.¹¹ X – xylose; G – glucose; L – galactose; F – fucose; OAc – O-acetylated oligosaccharide.(d) Error bars represent SD of technical replicates (n = 5). Different characters indicate significant differences (a; p < 0.05, one-way ANOVA with post-hoc Tukey HSD). The percentage of XyG O-acetylation (% XyG acet) was calculated based on the highest possible degree of XyG O-acetylation, taking only XyG oligosaccharides with O-acetylation sites into account (b, c; ^4,6). Error bars represent SD of technical replicates (n = 3).(e) Cell wall material (AIR) was extracted from a pool of 400–500 seedlings per genotype grown over 3 independent experiments (a, b, c).

OLIMP indicated that under control conditions, the overall proportions of O-acetylated xyloglucan were similar in dez and the wild type, and reduced by about 12% in rwa2-3 (Figure 1(b), % XyG acet). This suggested that in dez, the increase in total cell wall acetylation (see Figure 1(a)) could be a result of an increased proportion of xyloglucans in the cell wall resulting from a decrease in other components, or of an increased acetylation of other hemicellulose moieties not covered by OLIMP.

In seedlings cultivated in the dark under high-Zn conditions, in which photomorphogenic development is more evident in both the dez and the rwa2-3 mutant, we observed altered proportions of acetylated xyloglucans compared to seedlings cultivated under control conditions (Figure 1(c)). High-Zn dependent alterations were considerably more pronounced in dez, with a clear increase in overall xyloglucan acetylation by 75%. This increase was proportionally reflected in all macromolecular contexts analyzed here (XXLG/XLXG, XXFG, XLFG). Although dez and rwa2 both exhibited pronounced characteristics of photomorphogenesis in the dark under high-Zn cultivation conditions, xyloglucan O-acetylation was increased only in dez, but not in rwa2-3, when compared to the wild type. This suggested that enhanced levels of xyloglucan O-acetylation are unlikely to have a causal role in the photomorphogenic phenotype of dez.

Together, the data presented here and in our original manuscript highlight the complexity of the plant’s response to perturbations of the cell wall. Such perturbations can arise, for example, from mutations that affect cell wall modification or from nutritional imbalances, e.g. excess of a cation. The data presented here suggest that a decrease in the degree of pectin acetylation in dez by about 40% compared to the wild type¹ prompts complex compensatory changes in cell wall acetylation, which depend strongly on the externally supplied levels of Zn. Based on our data, rwa2-3, a mutant characterized as more generally and globally, but only mildly, defective in cell wall acetylation, appears to exhibit fewer compensatory changes in cell wall acetylation of generally lower magnitude, and notably, xyloglucan O-acetylation is generally decreased. Zn-enhanced photomorphogenesis in the dark, and its rescue by vsOG application, suggest that both the dez and the rwa2-3 mutant share a common aspect or consequence among their cell wall defects that is exacerbated by Zn and leads to photomorphogenic development.

Our results suggest that external Zn²⁺ supply affects in muro cell wall structure and biochemistry. Zn²⁺ may modulate the activity of apoplastic cell wall modifying enzymes, or alter wall properties or structure and thus the ability of enzymes to access their sites of action. Alternatively, Zn²⁺ may modulate the activity of RWA or TBL family proteins in the Golgi that act in the O-acetylation of various cell wall polymers, or modulate cellular biochemistry to affect the availability of acetyl-CoA. Further study of these mutants, including in vivo quantification of vsOGs will help clarify the mechanism by which skotomorphogenesis is maintained. Our results are of interest for the research community interested in cell wall structure and metabolism, cellulosic biofuels as well as for researchers studying light-dependent development and plant metal tolerance.

Funding Statement

This work was supported by the Deutsche Forschungsgemeinschaft (DFG, grants Kr1967/3-3 and Kr1967/15-1).